The long-term goal of this project will be to understand in detail the oligomerization of inward rectifier K+ channels with the ROMK2 and IRK1 channels as models. This will provide major insights into the structural organization of channels belonging to this important ion channel family and into the protein domains underlying their unique functional properties and pharmacology. Further, the research will likely yield invaluable information relating to the architecture and assembly of related voltage- gated K+ channel subunits with their complex predicted membrane topology and to other oligomeric membrane proteins in general. Potassium channels are fundamental to many processes: membrane potential maintenance and repolarization, regulation of action potential duration, potassium transport and homeostasis, transepithelial transport of other ions, cell volume regulation, and secretion of neurotransmitters and hormones. Voltage-gated K+ channel polypeptides oligomerize into tetrameric homomultimers or heteroligomers necessary for their numerous functions. Exhibiting even greater functional diversity are inward rectifier K+ channels (ATP-regulated, ATP-sensitive, steeply rectifying (classical), and G-protein coupled muscarinic K+ channels) which by sequence homology to the former are hypothesized to consist of tetramers. Little is known regarding the molecular steps in subunit oligomerization, interactions between protein segments, mechanisms for association and for determining specificity, heteromultimer formation, or subcellular location of assembly for these ion channels. We will (1) demonstrate the assembly of these channels in vitro and in cells, (2) determine the stoichiometry of these channels, (3) determine whether heteromultimeric channels can be formed by different alternatively-spliced subunits or by proteins from different subfamilies, and (4) identify protein segments and key residues involved in oligomerization and in maintaining specificity. Analysis of polypeptide sequences by deletion analysis, epitope probing, site-directed mutagenesis, and chimeric constructs will identify recognition domains. Oligomerization and localization will be studied by sucrose gradient centrifugation, immunoprecipitation, immunoblotting, confocal microscopy, and functional expression in Xenopus oocytes and baculovirus-infected Sf-9 cells.